MIM is essential for pore expansion and stabilization. (A) Schematic of the MIM gene structure and constructs expressed in transgenic lines. Exons (rectangles), introns (light gray bars; not to scale), UTRs (dark gray), regions encoding for the I-BAR domain (orange). The null allele MIM^null bears an internal deletion of exons 3–10. Isoform C (Iso-C) is the longest isoform and was used to generate MIM-Emerald/mScarlet; fluorescence tag (FT). (B) Mean frequency (%) of stalling, full collapse (FC), kiss-and-run (KAR), and membrane crumpling (MC) in MIM^+/Def, MIM^Null/Def, and MIM^KD (from Fig. 4 B, semi-transparent; presented for convenience) compared with WT (from Fig. 2 C, semitransparent; presented for convenience). MIM^+/Def is not significantly different from the WT. MIM^Null/Def resulted in an increase of kiss-and-run event frequency (49 ± 3% (****)) as opposed to MIM^KD, which resulted in an increase of full collapse event frequency (46 ± 16% (***)). SG expressing Glue-GFP and LifeAct-Ruby. Error bars = SEM. N (SGs) ≥ 3, n (events) ≥ 200. Statistical significance presented is compared with MIM^+/Def SGs. P values for MIM^Null/Def: ****(KAR) = 0.000031, ****(MC) = 0.00001. Two-tailed, unpaired multiple t tests corrected using the Holm-Sidak method. (C) Time-lapse sequence (SRRF intensity-projection) of representative LSVs from MIM^Null/Def undergoing kiss-and-run in SGs expressing the Glue-GFP (green) and LifeAct-Ruby (magenta) markers. Top: LSV (dashed line) undergoing kiss-and-run that recruited actin, deformed, and become displaced from the apical membrane (KAR w actomyosin). Bottom: Consecutive fusion events of the same LSV (at 00:00 and 12:48; KAR w/o actomyosin; asterisks) leading to actomyosin recruitment and membrane crumpling following the second fusion (13:52). Time mm:ss; relative to fusion. Scale bars = 1 µm. UAS expression in B and C driven by fkh-GAL4.